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International Journal of Nanomedicine 2017:12 473–486
International Journal of Nanomedicine Dovepress
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Open access Full Text article
http://dx.doi.org/10.2147/IJN.S123625
Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogenes
Yi Wang1
hui li1,2
Yan Wang1
hua li1
lijuan luo1
Jianguo Xu1
Changyun Ye1
1State Key Laboratory of Infectious Disease Prevention and control, National Institute for Communicable Disease control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, chinese center for Disease Control and Prevention, Changping, Beijing, 2Department of Microbiology, GuiZhou Medical University, Guiyang, Guizhou, People’s Republic of China
Abstract: Listeria monocytogenes, one of most problematic foodborne pathogens, is respon-
sible for listeriosis in both humans and animals and mainly transmitted through the food chain.
In this report, we propose a simple, rapid, and nearly instrument-free molecular technique using
multiple cross displacement amplification (MCDA) label-based gold nanoparticles lateral flow
biosensor (LFB) for specific, sensitive, and visual detection of L. monocytogenes. The MCDA-
LFB method was carried out at a constant temperature (61°C) for only 20 min during the reaction
stage, and then the amplification mixtures were directly detected by using LFB, eliminating the
use of an electrophoresis instrument, special reagents, or amplicon analysis equipment. The
whole procedure, from sample processing to result indicating, was finished within 1 h. The
analytical specificity of MCDA-LFB method was successfully determined by distinguishing
the target bacterium from other pathogens. The analytical sensitivity of the MCDA-LFB assay
was 10 fg of genomic templates per reaction in pure culture, which was in complete accordance
with MCDA by gel electrophoresis, real-time turbidity, and colorimetric indicator. The assay
was also successfully applied to detecting L. monocytogenes in pork samples. Therefore, the
rapidity, simplicity, and nearly equipment-free platform of the MCDA-LFB technique make
it possible for food control, clinical diagnosis, and more. The proof-of-concept assay can be
reconfigured to detect various target sequences by redesigning the specific MCDA primers.
Keywords: Listeria monocytogenes, gold nanoparticles, MCDA, MCDA-LFB
IntroductionListeria monocytogenes, a Gram-positive, facultative intracellular, foodborne bacteria
pathogen, is the causative agent of animals and human listeriosis.1 The opportunistic
organism is widespread in the natural environment, thus can easily contaminate various
raw and processed foods, such as meat, poultry, fish, vegetables, fermented sausages,
milk, and dairy products.2 L. monocytogenes has the ability to survive and grow even
under high concentrations of salt or bile, low temperature and pH, carbon starvation,
oxidative stress, and other adverse conditions.3 The occurrence of the target pathogen
is considered as a serious public threat because of its severity of the illness manifested
by gastroenteritis, septicemia, meningitis, spontaneous abortion, pneumonia, central
nervous system infections, etc.4
Human foodborne listeriosis was mainly linked to the consumption of
L. monocytogenes-contaminated food products and particularly associated with
ready-to-eat foods.5 The groups at high risk are pregnant women, newborn infants,
Correspondence: Changyun YeState Key Laboratory of Infectious Disease Prevention and control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, chinese center for Disease control and Prevention, Changbai Road 155, Changping, Beijing 102206, People’s Republic of ChinaTel +86 10 5890 0747Fax +86 10 5890 0748email yechangyun@icdc.cn
Journal name: International Journal of NanomedicineArticle Designation: Original ResearchYear: 2017Volume: 12Running head verso: Wang et alRunning head recto: MCDA-LFB for detection of Listeria monocytogenesDOI: http://dx.doi.org/10.2147/IJN.S123625
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Wang et al
older adults, and immunocompromised individuals.6 Despite
its low incidence, human listeriosis reminds of great public
health concern due to its high case fatality rate (20%–30%).7
Over the past two decades, numerous listeriosis outbreaks
were reported all over the world, such as Japan, New Zealand,
USA, France, England, Germany, and other European
countries.8 Hence, it is crucial to devise a rapid, highly sensi-
tive, and specific assay for L. monocytogenes detection.
Conventional techniques for the L. monocytogenes
detection are culture-based methods, which required trained
personnel and consist of multiple steps. Moreover, the slow
growth rate of the pathogenic bacteria is also a challenge
for detection by culture-based methods as the time to results
requires up to 7 days to produce reliable results.9,10 The
absence of high avidity antibodies has restricted the use of
antibody-based detection of the target pathogen, and as a
result, much effort has been devoted to nucleic acid-based
assays for efficient L. monocytogenes detection.11,12 In this
regard, polymerase chain reaction (PCR)-based identifica-
tion methods have been established for detecting the target
pathogens in enrichment cultures.2 However, these assays
required advanced and expertise laboratory system, were
expensive and not feasible in resource-challenged regions
of the world.
The growing use of molecular detection techniques has
emphasized simplicity and speed as key criteria for adoption
in field detection, “on-site” diagnosis, point-of-care testing
and more, and the isothermal amplification methodologies
were well-suited for these applications. Multiple cross dis-
placement amplification (MCDA) (Chinese IP Office Patent
Application CN201510280765.X) was a novel isothermal
amplification technique devised by our group and success-
fully applied to diagnostic detection of pathogens in food and
clinical samples.13,14 MCDA displayed unique advantages of
rapidity, simplicity, repeatability, sensitivity, and specificity,
generating amplicons from as few as three bacterial cells. The
MCDA products were detected by visual inspection of color
changes, by use of spectrophotometric apparatus to measure
turbidity, or by agarose gel electrophoresis. However, all
of the monitoring techniques are not specific to template
sequences, thus the nonspecific reaction products can cause
the false positive results. To overcome the technical short-
comings, further speedup and simplify the process of MCDA
technology, a novel lateral flow biosensor (LFB) was success-
fully devised and applied to detecting MCDA amplicons in
this study. Then, the MCDA assay combined with the novel
LFB (MCDA-LFB) was developed for simple, visual, and
rapid detection of L. monocytogenes. The analytical sensitivity
and specificity of L. monocytogenes-MCDA-LFB assay were
evaluated by pure cultures and the practical application was
also successfully verified by detecting the target pathogen
in pork samples.
Materials and methodsReagents and apparatusThe sheep anti-digoxigenin antibody (anti-Dig), the
streptavidin-immobilized 30 nm gold nanoparticles (SA-G),
and biotinylated bovine serum albumin (biotin-BSA) were
purchased from the Resenbio Co., Ltd. (Xi’an, People’s
Republic of China). The backing card, absorbent pad, nitro-
cellulose (NC) membrane, conjugate pad, and sample pad
were purchased from the Jie Yi Biotechnology Co., Ltd.
(Shanghai, People’s Republic of China). The Loopamp kits
and Loopamp™ Fluorescent Detection Reagent (FD) were
purchased from Eiken Chemical (Beijing, People’s Republic
of China). The DNA extraction kits (QIAamp DNA minikits;
Qiagen, Hilden, Germany) were purchased from Qiagen
(Beijing, People’s Republic of China).
Construction of gold nanoparticle-based dipstick biosensorThe LFB (4×6 mm) consists of an immersion pad, a conjugate
pad, an NC membrane, and an absorbent pad and assembled
on a plastic adhesive backing card orderly by overlapping
2 mm among them. A schematic diagram of the biosensor
was shown in Figure 1. The SA-G in 0.01 M phosphate-
buffered saline (PBS, pH 7.4) was deposited on the conjugate
pad. Anti-Dig (0.2 mg/mL) and biotin-BSA (2.5 mg/mL) in
0.01 M PBS (pH 7.4) were immobilized at the test line (TL)
and control line (CL) with each line separated by 5 mm,
respectively. The assembled cards were subsequently cut at
4 mm widths. All the biosensors were packaged in a plastic
bag containing a desiccant gel and dryly stored at the room
temperature until use.
Visual detection of MCDA products using the biosensorA 0.3 μL aliquot of MCDA products was deposited to the
sample application region of the biosensor. Then, a 100 μL
aliquot of running buffer (10 mM PBS, pH 7.4 with 1%
Tween 20) was also deposited to the sample application
region, and the biosensor allowed to absorb the whole run-
ning buffer. After 2 min, the MCDA product detection was
visualized in the form of red lines on the NC membrane.
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MCDA-LFB for detection of Listeria monocytogenes
Primer design for L. monocytogenes-MCDA-LFB assayTo design L. monocytogenes-specific MCDA primers,
a set of MCDA primers was designed by PrimerExplorer
V4 (Eiken Chemical, Japan) and primer software PRIMER
PREMIER 5.0 according to the specific lmo0733 gene
(GenBank accession no 985919).15 Blast analysis confirmed
that the MCDA primer set was specific for L. monocytogenes
strains. The details of primer design, primers sequences, loca-
tions, and modifications of MCDA primers were shown in
Table 1 and Figure 2. All of the oligomers were synthesized
and purified by TsingKe Biological Technology (Beijing,
People’s Republic of China) at high-performance liquid
chromatography purification grade.
Bacterial strains and genomic template preparationA total of 95 bacterial strains, including 50 strains of
L. monocytogenes, 23 strains of other Listeria species and
22 strains of non-Listeria bacteria, were used in the current
Figure 1 The outline of MCDA combined with LFB.Notes: (A) Schematic depiction of the new cross primer (CP1*) and amplification primer (D1*). (B) Outline of McDa with cP1* and D1*. (C) schematic illustration of the principle of lFB for visualization of McDa amplicons. (D) Interpretation of the results: I, positive (two red lines at the readout area) and II, negative (only the control line zone showed a red band). C1*, 5′-labeled with Dig when used in MCDA-LFB assay; CP1*, 5′-labeled with biotin when used in MCDA-LFB assay.Abbreviations: BSA, bovine serum albumin; Dig, digoxigenin; GNPs, gold nanoparticles; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification; NC, nitrocellulose; SA, streptavidin.
Table 1 The primers used in this study
Primers Sequences and modifications (5′–3′) Length
F1 aaggacTTTcgcaagaaga 19 ntcP1 aggaacTgacagTccTTgcTaTcaaacTgaaTgTggTgc 39 mercP1* Biotin-AGGAACTGACAGTCCTTGCTATCAAACTGAATGTGGTGC 39 merc1 aggaacTgacagTccTTgcT 20 ntc1* Dig-AGGAACTGACAGTCCTTGCT 20 ntD1 cccaTTTagagaTTgTTTgTc 21 ntr1 ggagaTTaacaTaTcggaaTc 21 ntr2 gaagTgcTTgaaacaccagT 20 ntD2 caaaggTTgaTgaTgTaaaagc 22 ntc2 cacTTTgcTTggagaaacTgTTaTg 25 ntcP2 cacTTTgcTTggagaaacTgTTaTgaagTTTaTaaTcTccagTTTTTcgg 50 merF2 ggcggTcTTTcTTTgagc 18 nt
Notes: C1*, 5′-labeled with Dig when used in MCDA-LFB assay; CP1*, 5′-labeled with biotin when used in MCDA-LFB assay. Abbreviations: LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification; mer, monomeric; nt, nucleotide.
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Wang et al
study (Table 2). All strains were stored in 15% (w/v) glycerol
broth at −70°C and then were refreshed three times on nutri-
ent agar plate at 37°C. According to the manufacture’s pro-
tocol, the genomic templates were extracted from all culture
strains using DNA extraction kits and were determined with
ultraviolet spectrophotometer (Nano drop ND-1000, Calibre,
Beijing, People’s Republic of China) at A260/280. The DNA
templates were stored under at −20°C before the templates
were used. The strains of L. monocytogenes serovar 1/2a
(EGD-e) were used for confirmation performance, optimal
temperature, and sensitivity analysis conducted in the study.
The genomic templates of L. monocytogenes serovar 1/2a
Figure 2 location and sequence of lmo0733 gene (Listeria monocytogenes-specific gene) used to design multiple cross displacement amplification primers.Notes: The nucleotide sequence of the sense strand of lmo0733 is shown. Right arrows and left arrows indicate sense and complementary sequences that were used.
Table 2 Bacterial strains used in this study
Bacteria Serovar Strain no (source of strain) No. of strains
Listeria monocytogenes 1/2a EGD-e 1ATCC51772 1Isolated strains (IcDc) 5
3a ATCC51782 1Isolated strains (IcDc) 5
1/2c slcc2372 1Isolated strains (IcDc) 5
3c Slcc2479 11/2b Slcc2755 1
Isolated strains (IcDc) 53b Slcc2450 17 slcc2482 1
NCTC10890 14a ATCC19114 1
Isolated strains (IcDc) 54c ATCC19116 1
Isolated strains (IcDc) 34b ATCC19115 1
Isolated strains (IcDc) 54d ATCC19117 1
Isolated strains (IcDc) 54e ATCC19118 1
Isolated strains (IcDc) 1Listeria ivanovii U ATCCBAA-678 1
U Isolated strains (IcDc) 5Listeria innocua U ATCCBAA-680 1
U Isolated strains (IcDc) 2Listeria grayi U ATCC25402 1
U Isolated strains (IcDc) 5Listeria seeligeri U ATCC35967 1
U Isolated strains (IcDc) 1
(Continued)
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MCDA-LFB for detection of Listeria monocytogenes
(EGD-e) were serially diluted (10 ng, 10 pg, 10 fg, 1 fg, and
0.1 fg per μL) and a 1 μL aliquot of each dilution was applied
for sensitivity examination of MCDA-LFB detection.
The standard MCDA assayTo determine the suitability of lmo0733-MCDA primers,
the MCDA reaction was performed as the standard MCDA
condition, which has been described in previous studies.13
Briefly, the MCDA reaction was conducted in 25 μL amplifi-
cation mixtures containing the following components: 0.8 μM
cross primers CP1*, 1.6 μM cross primer CP1, 2.4 μM cross
primer CP2, 0.8 μM each of amplification primers C1* and
C2, 1.2 μM each of amplification primers R1, R2, D1, and
D2, 0.4 μM each of displacement primers F1 and F2, 12.5 μL
2× reaction mix (Loopamp kits), 1.25 μL of Bst DNA poly-
merase (10 U), and 1 μL DNA template. The monitoring
techniques, including colorimetric indicator (FD), turbidi-
meters (LA-320C), gel electrophoresis, and LFB detection,
were applied to confirming the MCDA products.
Then, we evaluated the optimal amplification tempera-
ture of lmo0733-MCDA primers. The MCDA amplification
mixtures were carried out at a constant temperature ranging
from 60°C to 67°C for 1 h and then incubated at 85°C for
5 min to terminate the MCDA reactions. Mixtures with 1 μL
genomic template of Listeria ivanovii strain (L. ivanovii,
ATCCBAA-678) and Salmonella strain (ICDC-NPsa001)
were selected as negative controls, and mixtures with 1 μL
double distilled water were used as a blank control.
The analytical sensitivity of the L. monocytogenes-MCDA by four monitoring techniquesThe genomic templates of L. monocytogenes serovar 1/2a
(EGD-e) were serially diluted to verify the limit of detection
(LoD). The LoD was defined by genomic DNA amount of
the template. The analytical sensitivity of MCDA-LFB was
tested as described above, and was further confirmed by
colorimetric indicator (FD reagent), 2% agarose gel electro-
phoresis, and real-time turbidity analysis. Three replicates
of each dilution were determined to verify the analytical
sensitivity.
The analytical specificity of the L. monocytogenes-MCDA-LFB assayTo evaluate the analytical specificity of L. monocytogenes-
MCDA-LFB assay, the MCDA reactions were conducted
under the conditions described above with purely genomic
templates from 50 L. monocytogenes, 23 strains of other
Table 2 (Continued)
Bacteria Serovar Strain no (source of strain) No. of strains
Listeria welshimeri U ATCC35897 1U Isolated strains (IcDc) 5
Bacillus cereus U Isolated strains (IcDc) 2Enteropathogenic Escherichia coli U Isolated strains (IcDc) 1Enterotoxigenic E. coli U Isolated strains (IcDc) 1Enteroaggregative E. coli U Isolated strains (IcDc) 1Enteroinvasive E. coli U Isolated strains (IcDc) 1Enterohemorrhagic E. coli U EDL933 1Plesiomonas shigelloides U ATCC51903 1Shigella boydii U Isolated strains (IcDc) 1Shigella sonneri U Isolated strains (IcDc) 1Shigella flexneri U Isolated strains (IcDc) 1Enterobacter cloacae U Isolated strains (IcDc) 1Enterococcus faecalis U ATCC35667 1Yersinia enterocolitica U ATCC23715 1Streptococcus pneumoniae U Isolated strains (IcDc) 1Aeromonas hydrophila U ATCC7966 1Vibrio vulnificus U Isolated strains (IcDc) 1Proteus vulgaris U Isolated strains (IcDc) 1Vibrio fluvialis U Isolated strains (IcDc) 1Streptococcus bovis U Isolated strains (IcDc) 1Vibrio parahaemolyticus U aTcc17802 1Klebsiella pneumoniae U aTcc700603 1Salmonella enteric U ICDC-NPSa001 1
Abbreviations: ATCC, American Type Culture Collection; ICDC, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention; NCTC, National Collection of Type Culture; Slcc, Seeliger’s Special Listeria Culture Collection; U, unidentified serotype.
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Wang et al
Listeria species, and 22 strains of non-Listeria bacteria
(Table 2). The MCDA products were analyzed using
LFB detection. The evaluations were performed twice
independently.
Applicability of the L. monocytogenes-MCDA-LFB assayTo test the availability of the MCDA-LFB assay for detection
of L. monocytogenes, the analysis results of MCDA-LFB assay
for 61 pork samples (ground meat samples) obtained from
one meat wholesale market in Beijing were compared with
conventional PCR and culture-biotechnical detection results
for the same samples (Figure 3). The culture-biotechnical
detection of pork samples was conducted according to the
ISO 11290-1 standard method. In brief, 25 g of each pork
sample was added into 225 mL Half Fraser’s broth (Oxoid,
Hampshire, UK) and homogenized, followed by incubating
the Listeria enrichment broth at 30°C for 24 h. Then, aliquots
(100 μL) of Half Fraser’s broth were transferred to 10 mL
of Fraser’s broth and homogenized, followed by incubating
the broth at 37°C with shaking (250 rpm) for 48 h. A portion
(50 μL) of positive Fraser’s broth cultures was streaked onto
Polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol
Agar (PALCAM) agar plates (Oxoid), which were incubated
for 48 h at 37°C. The identification of L. monocytogenes
strains was confirmed by gram staining, characteristic
colony morphology, and various tests (motility, catalase,
oxidase CAMP test, indole, urease, and aesculin hydroly-
sis). A portion (1 mL) of the Fraser’s broth was subjected
to extract genomic DNA, which is used as a template in the
MCDA-LFB and PCR assay. A L. monocytogenes-PCR assay
has been developed in a report, which amplified a 113 bp
DNA fragment of lmo0733 and was employed to verify the
presence of target pathogens in pork samples.16
ResultsDevelopment of the MCDA-LFB assayA schematic illustration of the principle of MCDA-LFB
technique is presented in Figure 1. In the MCDA system, the
cross primers (CP1 or CP2) are modified at the 5′ end with
biotin and the amplification primers (C1 or C2) are labeled
at the 5′ end with Dig (Figure 1A). The new CP1, CP2, C1,
and C2 primers are termed as CP1*, CP2*, C1*, and C2*,
respectively. For clarity, the CP2* and C2* primers are not
shown in outline of MCDA reaction (Figure 1B). During the
reaction stage, the CP1* primer initiates MCDA reaction at
the P1s sequence of the target and this new strand is dis-
placed by upstream synthesis from the displacement primer
Figure 3 Schematic representation of MCDA-LFB method compared with PCR and standard culture methods.Abbreviations: BHI, brain heart infusion; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification; PCR, polymerase chain reaction.
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MCDA-LFB for detection of Listeria monocytogenes
F1 (step 1). Then, five primers (D1, C1*, R1, CP2, and F2)
anneal to the newly synthesized strand and the polymerase
(Bst) extended in tandem generating four distinct products
(step 2). The D1 product serves as the template for subse-
quent elongation and cycling steps (step 3, Cycle 1). The
C1* product is also used as the template by C1* and CP1*
primers, and enters a cyclic process (step 4, Cycle 2). In the
cycle, a larger amounts of double-labeled detectable products,
which contain Dig-labeled C1* primer and a biotin-labeled
CP1* primer, are successfully constructed. The details of the
amplification process for R1 and CP2 products (steps 5 and 6)
have been described in previous report.13 Furthermore, a
double-labeled detectable product (CP2*/C2* product),
which is similar to the detectable CP1*/C1* product, can be
formed when the C2 primer is modified with a Dig at the 5′ end and CP2 primer for biotin.
A schematic illustration of the principle of LFB for
visualization of MCDA amplicons is shown in Figure 1C.
The biosensor detects MCDA amplicons through specific
recognition of the Dig labels at the end of products, which
are produced by using Dig labeled primers (C1* primer). The
amplicons labeled with biotin bind streptavidin-conjugated
gold nanoparticles for visualization at the other end. The
MCDA products are deposited on the sample application
region of the biosensor, and then the running buffer is also
deposited on the sample application area. The running buffer
is used for rehydrating and releasing the dried detector
reagents (SA-G) from the conjugate region to the detection
zone by capillary migration. At the detection zone, the tar-
get amplicons are specifically captured by the immobilized
anti-Dig at the TL, which provides a visual response to
the presence/absence of the target amplicons. The detector
reagents rapidly accumulate in the reaction zone of the strip
through biotin/streptavidin interaction, resulting in a visual
red colored band at the TL. The proper functioning of the
strip is demonstrated by the CL formation, which contains
biotin-BSA that captured excess detector reagent. Therefore,
the appearance of two red lines in the readout region sug-
gests a positive test and only one red line in the CL suggests
a negative test (Figure 1D).
Validation and confirmation of L. monocytogenes-MCDA-LFB productsTo validate the feasibility of L. monocytogenes-MCDA
primers, the MCDA reactions were conducted in the
presence or absence of genomic DNA templates within
1 h at a constant temperature (61°C). The positive
L. monocytogenes-MCDA tube was visualized by naked eye
as green, whereas the negative and blank controls remained
light gray (Figure 4A). Agarose gel electrophoresis of the
MCDA products exhibited the typical ladder-like patterns in
the positive MCDA amplification but not in the negative and
blank controls (Figure 4B). It was also displayed that two
visible red bands (TL and CL) were observed in the positive
amplification and only the CLs were seen in negative
and blank controls (Figure 4C). These results indicated
that the L. monocytogenes-MCDA primer set was a good
candidate for development of the MCDA-LFB method for
L. monocytogenes detection.
The temperature optimization of the MCDA-LFB assay during the reaction stageAccording to the standard MCDA conditions, the MCDA
was carried out using the EGD-e DNA as the template at
the level of 10 pg genomic templates per reaction to exam-
ine the optimal assay temperature during the amplification
stage. Various amplification temperatures ranging from 60°C
to 67°C at 1°C intervals were compared for optimal assay
temperature. The real-time measurement of turbidity was
used for analyzing the MCDA amplifications and the typical
kinetics graphs corresponding to eight temperatures were
produced. As shown in Figure 5, the optimal results were
yielded from assay temperature of 60°C–63°C (Figure 5).
The reaction temperature of 61°C was selected for the rest
of MCDA-LFB tests conducted in this report.
Analytical sensitivity of MCDA-LFB technique in pure cultureTo test the LoD of L. monocytogenes-MCDA-LFB assay,
the assay was evaluated using serial dilutions (from 10 ng
to 0.1 fg per μL) of pure L. monocytogenes (EGD-e) DNA.
As shown in Figure 6A, the MCDA products were analyzed
by LFB detection, and the LoD of MCDA-LFB assay for
detecting lmo0733 gene was 10 fg of genomic templates per
reaction. As expected, the biosensor displayed clear visible
red lines for both TL and CL when the products came from
positive MCDA reactions, and only the CL were obtained
from negative MCDA amplifications, negative control, and
blank control.
Using the MCDA amplification by self-trail, the LoD
of the approaches was further confirmed by direct visual
inspection of the MCDA products with FD reagent and
electrophoresis in 2% agarose gels stained with ethidium
bromide. The positive amplifications by FD reagent were
indicated by a color change from light gray to green and
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Figure 4 Verification and detection of Listeria monocytogenes-MCDA products.Notes: (A) reaction products of L. monocytogenes-MCDA assay were visually detected by observation of the color change. (B) Agarose gel electrophoresis of L. monocytogenes-McDa products was shown. (C) lFB applied for visual detection of L. monocytogenes-MCDA products. Tube 1/lane 1/strip 1, positive amplification of L. monocytogenes strain (EGD-e); tube 2/lane 2/strip 2, negative control of Listeria ivanovii strain (ATCCBAA-678); tube 3/lane 3/strip 3, negative control of Salmonella strain (ICDC-NPSa001); tube 4/lane 4/strip 4, blank control (DW). Lane M, DNA maker DL 100.Abbreviations: DW, double distilled water; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification.
Figure 5 (Continued)
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MCDA-LFB for detection of Listeria monocytogenes
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Figure 6 Analytical sensitivity of MCDA-LFB assay using serially diluted genomic DNA with Listeria monocytogenes strain EGD-e.Notes: A total of 4 monitoring methods, including LFB (A), colorimetric indicator (FD) (B), gel electrophoresis (C), and real-time turbidity (D) were applied for analyzing the amplification products. The serial dilutions (10 ng, 10 pg, 10 fg, 1 fg, and 0.1 fg) of target templates were subjected to standard MCDA reactions. Strips (A)/tubes (B)/lanes (C)/turbidity signals (D) 1–8 represented the DNA levels of 10 ng, 10 pg, 10 fg, 1 fg, and 0.1 fg per reaction, negative control (10 pg of Listeria ivanovii genomic DNA), negative control (10 pg of Salmonella genomic DNA) and blank control (DW). The genomic DNA levels of 10 ng, 10 pg, and 10 fg per reaction produced the positive reactions.Abbreviations: BC, blank control; DW, double distilled water; FD, fluorescent detection reagent; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification.
Figure 5 Optimal reaction temperature for Listeria monocytogenes-MCDA primer sets.Notes: The standard McDa reactions for detection of L. monocytogenes were monitored by real-time measurement of turbidity and the corresponding curves of concentrations of DNA were marked in the figures. The threshold value was 0.1 and the turbidity of 0.1 was considered to be positive. Eight kinetic graphs (A–H) were generated at various temperatures (60°c–67°c, 1°C intervals) with target pathogens DNA at the level of 10 pg per reaction. The graphs from (A) to (D) show robust amplification.Abbreviation: MCDA, multiple cross displacement amplification.
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Wang et al
the positive results by electrophoresis were observed the
typical ladder-like patterns after 2% agarose gels electropho-
resis when compared with negative amplification (negative
reactions, negative control, and blank control; Figure 6B
and C). Moreover, the MCDA reactions were analyzed by
real-time turbidity detection and the decreasing concentra-
tions of pure L. monocytogenes DNA were displayed from
left to right. The analytical sensitivity of the two assays and
turbidity analysis was also 10 fg per reaction (Figure 6D).
These results indicated that the analytical sensitivity by FD
reagent, real-time turbidity, and agarose gel electrophoresis
detection for L. monocytogenes-MCDA amplifications was
conformity with biosensor analysis.
The time optimization of the MCDA-LFB assay during the reaction stageIn this report, we then examined the optimal duration of time
required for the MCDA-LFB assay during the reaction stage.
Various reaction times ranging from 10 to 25 min at 5 min
intervals were compared for optimal assay time according to
the standard MCDA conditions (Figure 7A, 7B, 7C and 7D).
The lowest genomic DNA level (10 fg of L. monocytogenes
templates per tube) exhibited two red lines when the reaction
only lasted for 20 min at 61°C (Figure 7C). A reaction time of
20 min was selected as the optimal time for the MCDA-LFB
assay during the reaction stage (Figure 7C). Therefore, the
whole procedure, including specimen (such as food sample)
processing (35 min), isothermal reaction (20 min), and result
reporting (5 min), could be finished within 60 min.
Figure 7 The optimal duration of time required for MCDA-LFB assay.Notes: Four different reaction times (A) 10 min, (B) 15 min, (C) 20 min, and (D) 25 min were tested and compared at 61°C. Strips 1–8 represent DNA levels of 10 ng of Listeria monocytogenes templates, 10 pg of L. monocytogenes templates, 10 fg of L. monocytogenes templates, 1 fg of L. monocytogenes templates, 0.1 fg L. monocytogenes templates per tube, negative control (Listeria ivanovii, 10 pg per reaciton), negative control (Salmonella, 10 pg per reaction), and blank control (DW). The best sensitivity was seen when the amplification lasted for 20 min (C).Abbreviations: DW, double distilled water; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification.
The analytical specificity of MCDA-LFB assayThe analytical specificity of the MCDA-LFB assay targeting
lmo0733 gene was evaluated with genomic DNA extracted
from 50 L. monocytogenes strains to 45 non-L. monocytogenes
strains (roughly 10 ng of genomic templates for each patho-
gen; Table 2). The positive results were seen in L. monocy-
togenes strains within a 20 min incubation period at 61°C.
As shown in Figure 8, two red bands, including TL and CL,
were produced in the LFB, and the sample was considered
as positive. By contrast, the detection was negative for the
non-L. monocytogenes strains and blank control after a
20 min incubation period. When only a band was observed
(CL) in the LFB, the sample was defined as negative. The
results demonstrated that the MCDA-LFB assay has a 100%
analytical specificity for L. monocytogenes detection.
evaluation of the L. monocytogenes-MCDA-LFB assay using pork samplesTo evaluate the feasibility of L. monocytogenes-MCDA-LFB
approach as a reliable surveillance tool for target pathogen in
food samples, 61 pork samples were analyzed by the culture-
biotechnical, PCR, and MCDA-LFB assays. The results were
shown in Figure 9 and summarized in Table 3. In case of
practical samples, 10 (16.4%) and 13 (21.3%) pork samples
were L. monocytogenes positive by PCR and MCDA-LFB
methods, respectively (Figure 9). The MCDA-LFB diag-
nostic accuracy obtained was 100% when compared to the
conventional culture-based assay (21.3%; Table 3). These
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MCDA-LFB for detection of Listeria monocytogenes
results suggested that the L. monocytogenes-MCDA-LFB
method developed here has higher detection ability when
compared to the traditional PCR approach.
DiscussionL. monocytogenes, recognized as an important foodborne
pathogen worldwide, is mainly transmitted through the food
chain and accounts for listeriosis – a severe illness of human
and animals with a high case fatality rate.17 The recent food
recalls and well-publicized outbreaks due to this bacterium
have increased the demand for more rapid, simple, specific,
and sensitive techniques for detection of L. monocytogenes
in various samples. Here, we proposed an approach using
MCDA combined with gold nanoparticles-based LFB
(MCDA-LFB) to offer a simple, rapid, highly sensitive,
and accurate method for detection of the target pathogen.
In the MCDA-LFB assay, gold nanoparticles were used
as reporter particles, because they are stable in both dried
and liquid forms and their colors do not fade after staining
on membranes.18 Gold nanoparticles also display distinct
physical and chemical attributes including high surface-
to-volume ratio, unique optoelectronic properties (surface
plasmon resonance), and good biocompatibility with DNA,
RNA, and proteins, and thus offer a well-suited platform
for colorimetric sensing of target analytes.19 Moreover, the
proof-of-concept strategy (MCDA-LFB) may be reconfig-
ured to detect a wide variety of nucleic acid sequences by
redesigning the specific MCDA primers.
To develop specific assays for L. monocytogenes detec-
tion, a large number of approaches, including PCR- and
loop-mediated isothermal amplification-based approaches,
were established for species-specific identification of this
bacterium, and the primer sequences were mainly derived
from two gene sequences: iap and hlyA gene.3 However,
the two target genes were not specific to L. monocytogenes,
iap gene was present in all six Listeria species and hlyA
gene for Listeria seeligeri, L. ivanovii, and L. monocyto-
genes.15 In this study, the MCDA primer sequences were
based on the species-specific gene (lmo0733), which was
unique to L. monocytogenes.15 The analytical specificity of
L. monocytogenes-MCDA-LFB assay was successfully veri-
fied with the genomic DNA extracted from 50 L. monocyto-
genes to 45 non-L. monocytogenes strains, and the positive
results were yielded in the assay of L. monocytogenes strains
but not for non-L. monocytogenes strains and blank control
(Figure 8). Thus, the proposed assay was highly selective to
target pathogen over other bacteria and could be as a potential
tool for screening L. monocytogenes with high specificity.
Figure 8 The specificity of MCDA-LFB assay for different strains.Notes: The MCDA reactions were carried out using different genomic DNA templates and were monitored by means of visual format. Biosensors 1–12, Listeria monocytogenes strains of serovar 1/2a (ATCC51772), 3a (ATCC51782), 1/2c (Slcc2372), 3c (Slcc2479), 1/2b (Slcc2755), 3b (Slcc2540), 7 (NCTC10890), 4a (ATCC19114), 4c (ATCC19116), 4b (ATCC19115), 4d (ATCC19117), and 4e (ATCC19118); biosensor 13–17, Listeria ivanovii (ATCCBAA-678), Listeria innocua (ATCCBAA-680), Listeria grayi (ATCC25402), Listeria seeligeri (ATCC35897), and Listeria welshimeri (ATCC35897); biosensors 18–39, Bacillus cereus, Enteropathogenic Escherichia coli, Enterotoxigenic E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, Enterohemorrhagic E. coli, Plesiomonas shigelloides, Shigella boydii, Shigella sonneri, Shigella flexneri, Enterobacter cloacae, Enterococcus faecalis, Yersinia enterocolitica, Streptococcus pneumonia, Aeromonas hydrophil, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Proteus vulgaris, Streptococcus bovis, Klebsiella pneumonia, and Salmonella enteric; and biosensor 40, blank control (DW).Abbreviations: ATCC, American Type Culture Collection; DW, double distilled water; LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification.
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Wang et al
Besides specificity, the analytical sensitivity was also
an important factor of the newly developed method in
practical application. Under the standard conditions, the
L. monocytogenes-MCDA-LFB assay proposed here was
capable of detecting as little as 10 fg of L. monocytogenes
genomic DNA per tube in pure culture (Figure 6). The
analytical sensitivity was further confirmed using gel elec-
trophoresis, FD reagent, and reaction-turbidity as indicators,
and the biosensor was as sensitive as gel electrophoresis,
FD reagent, and turbidity detection (Figure 6). Due to the
elimination of the requirement for electrophoresis, special
reagents, and detection apparatus, the MCDA-LFB method
was sometimes easy to determine, thus it was more suitable
than other MCDA-based techniques for rapid, simple, and
sensitive detection in various fields.
When the MCDA-LFB technique was applied to 61 pork
samples and compared with PCR and standard culture assays.
MCDA-LFB and culture-based methods could adequately
detect the bacterial loads in the practical samples, and
displayed higher detection sensitivity for target pathogen
compared to PCR assay (Table 3). Three samples were
confirmed to be positive by MCDA-LFB and culture-based
approaches, but negative by PCR detection. However,
as depicted in Figure 3, conventional culture-based assay was
time-consuming, complicated, and labor-intensive, and the
MCDA-LFB method was simpler and took only about 30 min
after DNA extraction. Consequently, the whole procedure,
including template preparation (35 min), isothermal reaction
(20 min), and result indicating (2 min), was finished within
60 min (Figure 3). By comparison among three assays, the
MCDA-LFB methodology is not only effective and accurate,
but also simpler and more rapid.
The test result of L. monocytogenes-MCDA-LFB pro-
vided direct visualization by naked eyes, which was very
easy to be interpreted. The interpretation of test results was
based on the appearance of red lines on the reaction zone. The
presence of two red lines (TL and CL) on the reaction region
indicated a positive result for L. monocytogenes, whereas
only a red line (CL) appeared in the reaction zone, indicat-
ing the negative results, negative controls, and blank control
(Figures 1C, 4 and 6–9). In previous reports, MCDA reactions
were analyzed using real-time turbidity, gel electrophoresis,
and colorimetric indicator (such as FD reagent).13 First,
the real-time turbidity detection suffered from background
interference, and the use of gel electrophoresis to analyze
MCDA products increased the risk of product degradation
and contamination. Second, the assessment of color change
with naked eye was potentially subjective, thus there was the
possibility that a sample was somewhat ambiguous to the
unaided eye when the concentration of target sequences was
low. More importantly, MCDA generated a complex mixture
of various amplicons due to use of 10 primers, thus these
monitoring techniques could not differentiate the nonspecific
and specific products.20 In the current study, we purposefully
modified the MCDA assay by using two primers (CP1/C1 or
CP2/C2) labeled with digoxigenin and biotin. This allowed
Figure 9 MCDA-LFB assay for the detection of Listeria monocytogenes in raw meat samples.Notes: A total of 61 pork samples were analyzed using L. monocytogenes-MCDA-LFB assay, and 13 samples (samples 9, 14, 16, 25, 28, 30, 31, 36, 39, 48, 50, 53, and 58) were L. monocytogenes positive.Abbreviations: LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification.
Table 3 Comparison of culture-biotechnical, MCDA-LFB, conve-ntional Pcr, for the detection of Listeria monocytogenes in raw meat samples
Detection methods Pork samples (n=61)
Positive Negative
culture 13 48MCDA-LFB 13 48Pcr 10 51
Abbreviations: LFB, lateral flow biosensor; MCDA, multiple cross displacement amplification; PCR, polymerase chain reaction.
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MCDA-LFB for detection of Listeria monocytogenes
labeled MCDA amplicons to be specifically analyzed using
the biosensor housed inside of a plastic, leak-proof device,
without the need for a turbidimeter, an electrophoresis instru-
ment, or colorimetric indicator.
Comparing with PCR-based assays, MCDA-LFB
method does not require accurate and rapid temperature
control during the reaction stage, obviating the need for
a sophisticated instrument for thermal management, and
operates between temperatures ranging from 60°C to 67°C
without losing amplification efficiency. Only a simple water
bath or heater that maintains a constant temperature is
sufficient, and this permits simplification of apparatus and
reduction of costs. A wide variety of portable user-friendly
instruments adapted for MCDA reaction exist, the dry block
heater (HDT-100C, HengAo, Tianjing, People’s Republic
of China) being one example. The portable (18×22 cm),
battery-powered, $600 USD device supports 96 MCDA
reactions per assay. Currently, the MCDA amplification can
be performed using the commercial isothermal amplification
kits (such as NEB Warmstart kits and Eiken Loopamp kits),
and a MCDA reaction costs ~$3.5 USD. The cost of LFB
devised in this study is estimated to be $2 USD per test.
Combined with the elimination of labor costs because of
the requirements for trained personnel in a certified labora-
tory, the MCDA-LFB assay becomes more cost-effective.
Therefore, we deem that a MCDA-LFB assay would cost
~$6 USD per disposable and $600 USD or less for a dedi-
cated apparatus.
ConclusionIn this study, we reported a rapid, simple, and nearly
instrument-free molecular technique, which incorporated
MCDA coupled with gold nanoparticle-based LFB for visual,
specific, and sensitive detection of nucleic acid sequence.
The proof-of-concept strategy may be reconfigured to detect
various target sequences by redesigning the specific MCDA
primers. The MCDA-LFB technology devised here obviated
the thermal cycle steps, and incubating at a constant tempera-
ture using a simple block heater or water bath was sufficient to
replicate target sequences to detectable levels within a short
time (20 min for the amplification stage). Moreover, the use
of the newly devised biosensor provided an objective and
easily interpretable readout of approach’s results, alleviat-
ing the use of special reagents and expensive equipment.
As a proof of concept, the pathogen (L. monocytogenes),
which is responsible for severe listeriosis in both humans
and animals, was detected by MCDA-LFB to demonstrate
the capability of target identification. Our data showed that
L. monocytogenes-MCDA-LFB assay was advantageous
on quick results, easy operation, modest equipment require-
ments, high specificity and sensitivity, and cost and energy
efficiency, thus could achieve the clinical diagnosis, infection
control, and food hygiene inspection. Hence, these advan-
tages made the MCDA-LFB system possible for point-of-care
testing, genetic testing in resource-poor settings, and rapid
testing of environment samples and food products.
AcknowledgmentsWe acknowledge the financial supports of the grants (Mega
Project of Research on the Prevention and Control of HIV/
AIDS, Viral Hepatitis Infectious Diseases 2013ZX10004-
101 to Changyun Ye) from the Ministry of Science and
Technology, People’s Republic of China, and grant
(2015SKLID507 to Changyun Ye) from State Key
Laboratory of Infectious Disease Prevention and Control,
China CDC.
Author contributionsYW and CY conceived and designed the experiments; YW,
HL, YW, HL, and LL performed the experiments; YW and
HL analyzed the data; YW, HL, YW, HL, LL, JX, and CY
contributed reagents/materials/analysis tools; YW, JX, and
CY wrote the paper. All authors contributed toward data
analysis, drafting and revising the paper and agree to be
accountable for all aspects of the work.
DisclosureYi Wang and Changyun Ye have filed for a patent from
the State Intellectual Property Office of the People’s
Republic of China, which covers the novel method and
sequences included in this manuscript (application number:
CN201610892015.1). The other authors report no conflicts
of interest in this work.
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